Hybridization chain reaction

ABSTRACT

The present invention relates to the use of nucleic acid probes to identify analytes in a sample. In the preferred embodiments, metastable nucleic acid monomers are provided that associate in the presence of an initiator nucleic acid. Upon exposure to the initiator, the monomers self-assemble in a hybridization chain reaction. The initiator nucleic acid may be, for example, a portion of an analyte to be detected or may be part of an initiation trigger such that it is made available in the presence of a target analyte.

REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 60/556,147, filed Mar. 25, 2004

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the use of nucleic acidprobes to identify analytes in a sample and more particularly to the useof metastable nucleic acid monomers that self-assemble upon exposure toa target analyte.

2. Description of the Related Art

Single stranded DNA is a versatile material that can be programmed toself-assemble into complex structures driven by the free energy of basepair formation. Synthetic DNA machines can be powered by stranddisplacement interactions initiated by the sequential introduction ofauxiliary DNA fuel strands. Typically, various DNA strands begin toassociate as soon as they are mixed together. Catalytic fuel deliveryproves a conceptual approach to powering autonomous DNA machines bystoring potential energy in loops that are difficult to accesskinetically except in the presence of a catalyst strand.

SUMMARY OF THE INVENTION

Metastable nucleic acid monomers can be made that self-assemble uponexposure to an initiator, such as a target analyte. In one aspect of theinvention, methods are provided for using such monomers to detect ananalyte in a sample. The sample is contacted with a first metastablemonomer comprising an initiator complement region and a secondmetastable monomer comprising a region that is complementary to aportion of the first monomer. The monomers may be, for example, hairpinnucleic acid structures comprising a loop region and a duplex region.

The first and second monomers polymerize in the presence of aninitiator. Preferably, hybridization of the initiator to the initiatorcomplement region of the first monomer initiates polymerization.Polymerization continues until the supply of one of the monomers isexhausted. The identification of polymers comprising the first andsecond monomers is indicative of the presence of the analyte in thesample. Polymers may be identified, for example, by gel electrophoresis

The initiator is preferably a nucleic acid. In some embodiments, theanalyte comprises the initiator. In other embodiments the sample isadditionally contacted with an initiation trigger. The initiationtrigger preferably comprises the initiator and a binding molecule, suchas an aptamer, that is able to specifically recognize the analyte ofinterest. The initiator is able to hybridize to the first monomer,triggering polymerization, when the binding molecule is bound by theanalyte.

In one embodiment the analyte is a nucleic acid that is associated witha pathogen, such as HIV. The sample may be a biological sample from apatient.

According to another aspect of the invention, methods are provided forforming a structure comprising hybridized nucleic acid monomers. Aninitiator comprising a nucleic acid initiation region is provided. Theinitiator may be a single stranded nucleic acid. In some embodiments,the initiator comprises a recognition molecule, such as an aptamer. Inone embodiment the initiator is an analyte to be detected in a sample,such as a biological sample obtained from a patient.

A first metastable nucleic acid hairpin monomer is provided comprising aduplex region, a first loop region and an initiator complement regionthat is substantially complementary to the initiation region. The loopregion of the first monomer preferably comprises from about 3 to about30 nucleotides.

A second metastable nucleic acid hairpin monomer is provided thatcomprises a second duplex region and a propagation region that issubstantially complementary to the initiator complement region of thefirst monomer. The propagation region may be, for example, a second loopregion.

The first and second monomers are able to hybridize in a chain reactionto form a polymer structure upon hybridization of the initiator to thefirst monomer. The polymer structure may be detected, for example, by amethod selected from the group consisting of gel electrophoresis,capillary electrophoresis, mass spectrometry, light scatteringspectroscopy, colorimetry and fluorescent spectroscopy.

In some embodiment, a third and fourth metastable nucleic acid monomerare provided. The third monomer preferably comprises a region that iscomplementary to a portion of the first and/or second monomer and thefourth monomer preferably comprises a region complementary to a portionof the third monomer.

In a further aspect of the invention, a kit is provided for detecting ananalyte in a sample. The kit preferably comprises a first metastablenucleic acid monomer comprising an initiator complement region and asecond metastable nucleic acid monomer comprising a propagation region.In one embodiment the monomers are hairpin nucleic acid monomers. Thekit may also comprise an initiation trigger. The initiation triggerpreferably comprises a region that is substantially complementary to theinitiator complement region of the first monomer. The initiation triggermay also comprise a target recognition molecule, such as an aptamer thatis able to specifically bind the analyte to be detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates one embodiment of an HCR system. Eachletter represents a segment of nucleic acids. Letters marked with a *are complementary to the corresponding unmarked letter.

FIG. 1A shows two hairpins, labeled H1 and H2, that are metastable inthe absence of initiator I. The hairpins comprise sticky ends ‘a’ and‘c*’, respectively. Potential energy is stored in the hairpin loops.

FIG. 1B shows how a single initiator strand ‘I’ can nucleate or bind tothe sticky end of H1 and displace one arm to open the hairpin. Thisfrees up the bases that were trapped in the hairpin, allowing them toperform a similar displacement reaction on H2.

As illustrated in FIG. 1C, the newly exposed c region of H1 nucleates atthe sticky end of H2 and opens the hairpin to expose a region on H2 (a*)that is identical in sequence to the initiator I. As a result, each copyof I can propagate a chain reaction of hybridization events betweenalternating H1 and H2 hairpins to form a nicked double helix, therebyamplifying the signal of initiator binding. The process can continueuntil the monomers (H1 and H2) are exhausted. At each step, energy isgained from the hybridization of ‘a’ or ‘c’. The reactions diagrammed inFIG. 1 have been successfully carried out and are summarized in FIG. 1D.

FIG. 1D illustrates the results of an HCR reaction and the effect ofinitiator concentration on amplification. Lanes 2-7: Six differentconcentrations of initiator were used (0.00, 10.00, 3.20, 1.00, 0.32 and0.10 μM, respectively) in a 1 μM mixture of H1 and H2. Lanes 1 and 8were DNA markers with 100-bp and 500-bp increments respectively.

FIG. 1E illustrates HCR kinetics monitored by substituting afluorescently labeled base in the sticky end of an HCR monomer. Here2-aminopurine (2AP) was substituted for A (base 3) in the sticky end ofH1. The hairpin monomers H1 and H2 did not hybridize prior to triggeringby initiator ((H1^(2AP)+1.2×H2 for 24 hours+0.5× initiator), red). Thesame quenched baseline was achieved without HCR by adding excessinitiator to H1^(2AP) in the absence of H2 (H1^(2AP)+4.0× initiator,green). Addition of insufficient initiator to H1^(2AP) provided onlypartial quenching (H12AP+0.5× initiator (blue), demonstrating that HCR,and not initiator alone, was responsible for exhausting the supply ofH12AP monomer.

FIG. 2 illustrates an embodiment in which HCR utilizing two monomerpairs produces quadratic signal amplification. FIG. 2A illustrates twohairpin monomers Q1 and Q2 that are metastable in the absence ofinitiator IQ. As shown in FIG. 2B, binding of IQ leads to a stranddisplacement interaction that exposes sticky end fe*b*. This singlestranded region then nucleates at the f* sticky end of Q2, and asubsequent branch migration exposes segments cb*d* and e* as shown inFIG. 2C. The d*e* region initiates the next Q1 molecule, leading toamplification in one direction, while the exposed cb* region initiates asecond HCR reaction involving monomers H1 and H2 (FIG. 1). Asillustrated in FIG. 2D, the resulting branched polymer has a Q1/Q2 mainchain with H1/H2 side chains branching off at each Q2 segment.

FIG. 3 illustrates a pair of monomers (E1 and E2) that can be used incombination with at least one other pairs of monomers to achieveexponential amplification by HCR. In the presence of cb*, E1 and E2 forma linear chain that includes periodic single stranded d*e* regions. Theinitiator sequence for E1/E2 matches the periodic single stranded regionproduced by Q1/Q2 and vice versa. Consequently, a mixture of Q1, Q2, E1and E2 plus either initiator (cb* or d*e*) will lead to the formation ofa structure in which each branch of the polymer is itself a branchedpolymer. Sustained growth will ultimately decrease to cubicamplification.

FIGS. 4A-E illustrate another embodiment for HCR with exponentialgrowth. Eight different strands are used in this embodiment. Strand one(1) is the ‘hub’ of the system and has an exposed, single-strandedregion joining two hairpins (FIG. 4A). When the initiator (2) binds, itcreates a long helix with one sticky end on each side. The two stickyends generated by the initiated ‘hub’ bind with strands (3) and (6),respectively (FIGS. 4B and C). Next, auxiliary strands (4) and (7) bindto previously protected bulge loops (FIGS. 4D and E), and expose twohairpin regions. These hairpins then bind to strands (5) and (8),respectively, to generate sticky ends similar to the initiator molecule(2). Thus, each initiator produces two new initiators, leading toexponential growth. As a side note, two subsets of strands (1,2,3,4,5)and (1,2,6,7,8) produce linear systems in the absence of the otherstrands.

FIG. 5A illustrates an aptamer HCR trigger mechanism for the detectionof ATP. Binding of the DNA aptamer to ATP induces a conformation changethat exposes a sticky end.

FIG. 5B shows an agarose gel demonstrating ATP detection via HCR.

FIG. 6 illustrates a self-complementary hairpin with an interior loopthat is a double helix (dotted lines). DNA hairpin can also exist as adimer with an interior loop. One possible concern is that the interiorloops may be easier to invade than the corresponding hairpins. Toprevent this side reaction, self-complementary hairpins would convertthe interior loop to a simple double helix (dotted lines). However, thisadded precaution may not be necessary.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Hybridization Chain Reaction (HCR) is a novel method for the triggeredhybridization of nucleic acid molecules starting from metastable monomerhairpins or other metastable nucleic acid structures. Dirks, R. andPierce, N. Proc. Natl. Acad. Sci. USA 101(43): 15275-15278 (2004),incorporated herein by reference in its entirety. HCR does not requireany enzymes and can operate isothermally.

In one embodiment of HCR, two or more metastable monomer hairpins areused. The hairpins preferably comprise loops that are protected by longstems. The loops are thus resistant to invasion by complementarysingle-stranded nucleic acids. This stability allows for the storage ofpotential energy in the loops. Potential energy is released when atriggered conformational change allows the single-stranded bases in theloops to hybridize with a complementary strand, preferably in a secondhairpin monomer.

Each monomer is caught in a kinetic trap, preventing the system fromrapidly equilibrating. That is, pairs of monomers are unable tohybridize with each other in the absence of an initiator. Introductionof an initiator strand causes the monomers to undergo a chain reactionof hybridization events to form a nicked helix (see FIGS. 1A-C). HCR canbe used, for example, to detect the presence of an analyte of interestin a sample. This and other applications are discussed in more detailbelow.

Definitions

“Nucleic Acids” as used herein means oligomers of DNA or RNA. Nucleicacids may also include analogs of DNA or RNA having modifications toeither the bases or the backbone. For example, nucleic acid, as usedherein, includes the use of peptide nucleic acids (PNA). The term“nucleic acids” also includes chimeric molecules.

The term “sticky end” refers to a nucleic acid sequence that isavailable to hybridize with a complementary nucleic acid sequence. Thesecondary structure of the “sticky end” is such that the sticky end isavailable to hybridize with a complementary nucleic acid under theappropriate reaction conditions without undergoing a conformationalchange. Typically the sticky end is a single stranded nucleic acid.

“Monomers” are individual nucleic acid oligomers. Typically, at leasttwo monomers are used in hybridization chain reactions, although three,four, five, six or more monomers may be used. In some embodiments morethan two monomers are utilized, such as in the HCR systems displayingquadratic and exponential growth discussed below. Typically each monomercomprises at least one region that is complementary to at least oneother monomer being used for the HCR reaction.

A first monomer in a monomer pair preferably comprises an initiatorcomplement region that is complementary to a portion of an initiatormolecule. The initiator complement region is preferably a sticky end.Binding of the initiator to the initiator complement region begins anHCR reaction.

In addition, the second monomer preferably comprises a propagationregion that is able to hybridize to the initiator complement region ofanother monomer, preferably another copy of the first monomer, tocontinue the HCR reaction begun by the initiator. The propagation regionmay be, for example, the loop region of a hairpin monomer as describedbelow. In one embodiment the propagation region on the second monomer isidentical to the portion of the initiator that is complementary to theinitiator complement region of the first monomer.

The propagation region on the second monomer is preferably onlyavailable to interact with the initiator complement region of the firstmonomer when an HCR reaction has been started by the initiator. That is,the propagation region becomes available to hybridize to the initiatorcomplement region of another monomer when one copy of the first monomerhas already hybridized to a second monomer, as discussed in more detailbelow.

Preferred monomers are “metastable.” That is, in the absence of aninitiator they are kinetically disfavored from associating with othermonomers comprising complementary regions. “HCR” monomers are monomersthat are able to assemble upon exposure to an initiator nucleic acid toform a polymer.

As used herein, “polymerization” refers to the association of two ormore monomers to form a polymer. The “polymer” may comprise covalentbonds, non-covalent bonds or both. For example, in some embodiments twospecies of monomers are able to hybridize in an alternating pattern toform a polymer comprising a nicked double helix. The polymers are alsoreferred to herein as “HCR products.”

An “initiator” is a molecule that is able to initiate the polymerizationof monomers. Preferred initiators comprise a nucleic acid region that iscomplementary to the initiator complement region of an HCR monomer.

Monomers

Two or more distinct species of nucleic acid monomers are preferablyutilized in an HCR reaction. Each monomer species typically comprises atleast one region that is complementary to a portion of another monomerspecies. However, the monomers are designed such that they arekinetically trapped and the system is unable to equilibrate in theabsence of an initiator molecule that can disrupt the secondarystructure of one of the monomers. Thus, the monomers are unable topolymerize in the absence of the initiator. Introduction of an initiatorspecies triggers a chain reaction of alternating kinetic escapes by thetwo or more monomer species resulting in formation of a polymer. In theexamples below, the two hairpin monomers polymerize in the presence ofan initiator to form a nicked, double helix.

In a preferred embodiment, two or more monomer species are employed thathave a hairpin structure. The hairpin monomers preferably comprise loopsprotected by long stems. In other embodiments, monomers with a differentsecondary structure are provided. However, the secondary structure ispreferably such that the monomers are metastable under the reactionconditions in the absence of an initiator nucleic acid. In the presenceof an initiator, the secondary structure of a first monomer changes suchthat it is able to hybridize to a sticky end of a second monomerspecies. This in turn leads to a change in the secondary structure ofthe second monomer, which is then able to hybridize to another firstmonomer and continue the process. In this way, once a single copy of thefirst monomer interacts with a single copy of the initiator, a chainreaction is produced such that the monomers are able to assemble into apolymer comprising alternating monomer species.

A number of criteria can be used to design the monomers to achieve thedesired properties. These include, for example and without limitation,sequence symmetry minimization, the probability of adopting the targetsecondary structure at equilibrium, the average number of incorrectnucleotides at equilibrium relative to the target structure, andhybridization kinetics.

Monomers can be synthesized using standard methods, includingcommercially available nucleic acid synthesizers or obtained fromcommercial sources such as Integrated DNA Technologies (Coralville,Iowa).

In some embodiments, monomers are derivitized with a compound ormolecule to increase the molecular weight of the polymer resulting fromHCR. Preferably they are derivitized at a location that does notinterfere with their ability to hybridize. In other embodiments monomerscomprise a fluorophore or calorimetric compound that allows theresulting polymers to be visualized.

In preferred embodiments, at least two hairpin monomers are utilized asillustrated in FIG. 1A. The monomers each preferably comprise a stickyend (a and c*, respectively), a first complementary segment (b and b*,respectively), a loop segment (c and a*, respectively), and a secondcomplementary segment (b and b*, respectively). The first and secondcomplementary segments are also referred to as “stems” and together forma duplex region.

The first monomer (H1) preferably comprises a sticky end a that iscomplementary to a first nucleic acid portion a* of an initiator (I;FIG. 1B). This sticky end is referred to herein as the “initiatorcomplement region.” The initiator may be, for example, an analyte ofinterest, or a nucleic acid that is able to contact the first monomeronly in the presence of an analyte of interest, as discussed in moredetail below.

The second monomer (H2) preferably comprises a sticky end c* that iscomplementary to a portion of the first monomer that becomes accessibleupon initiator binding. Preferably the sticky end c* is complementary tothe loop segment c of the first monomer (FIG. 1A). The loop segment c ofthe first monomer is preferably not available to hybridize with stickyend c* of the second monomer in the absence of initiator.

The first and second complementary segments (b and b*) in the first andsecond monomers are typically substantially identical. That is, thefirst complementary segment b of the first monomer (H1) is able tohybridize to the second complementary segment b* of the second monomer(H2).

The first complementary segment of each monomer is also able tohybridize to the second complementary segment of the same monomer toform the hairpin structure. For example, as shown in FIG. 1A, the firstmonomer (H1) comprises a first complementary segment b that is able tohybridize to the second complementary segment b*. In the absence of aninitiator, the first and second complementary segments of each monomerare generally hybridized to form a duplex region of the metastablemonomer.

Preferably, the first complementary segment b of the first monomer isalso complementary to a portion b* of the initiator, such that uponhybridization of the initiator region a* to the sticky end a (theinitiator complement region) of the first monomer H1, one arm of thehairpin structure is displaced. This opens the hairpin and allowsbinding of the first complementary segment b to the second portion b* ofthe initiator strand (FIG. 1B).

The loop segment c of the first monomer is also exposed by the openingof the hairpin and is able to bind to the sticky end c* of the secondmonomer H2, as illustrated in FIG. 1C. This opens the second monomerhairpin H2 and the second complementary segment b* of the first monomeris able to hybridize to the first complementary segment b of the secondmonomer H2.

This leaves the loop region a* and first complementary region b* of thesecond monomer H2 exposed (FIG. 1C). The sticky end a of another firstmonomer (H1) species is able to bind to the exposed loop region a* ofthe second monomer H2, thus opening the H1 hairpin and continuing theprocess described above. Because the loop region a of the second monomeracts as an initiator on a second H1 monomer and allows the process tocontinue in the absence of further initiator, it is referred to as thepropagation region.

At each step, energy is gained from the hybridization of the the stickyend of the monomer. The result is a nicked, double helix polymercomprising alternating H1 and H2 fragments. This process preferablycontinues in a chain reaction until all of one or both of the monomerspecies is used up, or the reaction is stopped by some other mechanism.If desired, the nicks in the nucleic acid polymer structures that resultfrom HCR can by ligated (for example, using T4 DNA ligase).

Because of the self-propagating nature of the reaction, each copy of theinitiator species can begin the chain reaction. Further, as long asthere is a fixed supply of monomers the average molecular weight of theresulting polymers is inversely related to the initiator concentration,as can be seen in FIG. 1D.

The length of the loop, stem and sticky ends of the monomers can beadjusted, for example to ensure kinetic stability in particular reactionconditions and to adjust the rate of polymerization in the presence ofinitiator. In one preferred embodiment the length of the sticky ends isthe same as the length of the loops. In other embodiments the stickyends are longer or shorter than the loops. However, if the loops arelonger than the sticky ends, the loops preferably comprise a region thatis complementary to the sticky end of a monomer.

In some preferred embodiments the length of the loops is short relativeto the stems. For example, the stems may be two or three times as longas the loops.

The loop regions are preferably between about 1 and about 100nucleotides, more preferably between about 3 and about 30 nucleotidesand even more preferably between about 4 and about 7 nucleotides. In oneembodiment the loops and sticky ends of a pair of hairpin monomers areabout 6 nucleotides in length and the stems are about 18 nucleotideslong.

Other refinements to the system stabilize the monomer hairpins to helpprevent HCR in the absence of an initiator. This can be achieved, forexample, via super-stable hairpin loop sequences (Nakano et al.Biochemistry 41:14281-14292 (2002)), with ostensible structural featuresthat could further inhibit direct hybridization to the hairpin. In otherembodiments hairpin loops are made to be self-complementary at theirends. This self-complementarity “pinches” the hairpin loops, making themshorter. However, if the reactive sticky ends of each monomer arecomplementary to the loop regions on the opposite monomer, as describedabove, they will have a slight propensity to close up, thereby slowingdown the reaction. This feature can be utilized if a slower reaction isdesired. Completely self-complementary hairpins can also be used, forexample if the monomer hairpins are forming dimers with interior loopsthat are more easily invaded than their hairpin counterparts. FIG. 6illustrates a self-complementary hairpin with an interior loop that is adouble helix.

Reaction conditions are preferably selected such that hybridization isable to occur, both between the initiator and the sticky end of a firstmonomer, and between the complementary regions of the monomersthemselves. The reaction temperature does not need to be changed tofacilitate the hybridization chain reaction. That is, the HCR reactionsare isothermic. They also do not require the presence of any enzymes.

Variations

There are many possible variations to HCR that may improve its speed,stability and ability to amplify chemical signals. The systemillustrated in FIG. 1 and discussed above exhibits linear growth inresponse to initiator. However, increasing the rate of polymer growthcan enhance the ability to detect the presence of low copy numbertargets, such as a single target molecule in a large test volume. Forexample, monomers can be designed to undergo triggered self-assemblyinto branched structures exhibiting quadratic growth or dendriticstructures exhibiting exponential growth. The exponential growth islimited by the available space such that it decreases to cubicamplification as the volume around the initiator fills. However, ifchain reactions products are able to dissociate, exponential growth canbe maintained until the supply of monomers is exhausted.

In order to achieve non-linear growth, 3 or more HCR monomers can beused. In preferred embodiments at least 4 HCR monomers are used. In someembodiments, at least one monomer in a primary monomer pair incorporatea trigger nucleic acid segment that is complementary to the exposedsticky end of one of the monomers from a secondary set of HCR monomers.Upon exposure to the nucleic acid that is to be detected, the set ofprimary monomers undergoes HCR to form a polymer with a periodic singlestranded trigger region. Thus the trigger nucleic acid is exposed,leading to a polymerization chain reaction in the secondary set ofmonomers. In other embodiments, both the primary and secondary set ofmonomers includes a trigger segment, such that exponential growth isachieved. Exemplary schemes are presented in FIGS. 2 and 3 for achievingquadratic and exponential growth, respectively.

In one embodiment, one of a first pair of monomers comprises a bulgeloop. Upon polymerization, a single stranded region results from thepresence of the bulge loop. The bulge loop segment is preferablycomplementary to the sticky end of one of a second pair of HCR monomers.Thus, upon exposure to the initiator, the first pair of monomersundergoes HCR to form a polymer with a single stranded region that actsto trigger polymerization of the second pair of monomers. FIGS. 2A-Cdepict such a quadratic amplification scheme. Monomers Q1 and Q2interact with hairpin monomers H1 and H2 (FIG. 1) after initiation by IQto form the branched polymer schematically illustrated in FIG. 2D.

Q1 and Q2 (FIG. 2 a) are metastable in the absence of the initiator IQ.IQ binds to Q1 and a subsequent strand displacement interaction exposessegments f, e* and b* as shown in FIG. 2B. This single-stranded regioncontacts sticky end f* of Q2 and a subsequent branch migration exposessegments c, b*, d* and e*. Segment d* then interacts with another copyof Q1 at sticky end d, causing the hairpin to open up such that e* canalso hybridize. At the same time, the exposed c segment initiates alinear HCR reaction with hairpins H1 and H2 (not shown). The resultingbranched polymer has a main chain comprising alternating Q1 and Q2segments and H1/H2 side chains branching off at each Q2 segment.

In a further embodiment, exponential growth is achieved in response toan initiator by combining two or more pairs of monomers. For example,monomer pair Q1 and Q2 (FIG. 2) can be used in conjunction with monomersE1 and E2 (FIG. 3) to obtain exponential growth in response to aninitiator. In the presence of nucleic acid segment cb*, E1 and E2 form alinear chain that includes periodic single stranded d*e* regions. Bydesign, the initiator sequence for E1/E2 matches the periodic singlestranded region produced by Q1/Q2 and vice versa. Consequently, amixture of Q1, Q2, E1 and E2 monomers in the presence of initiator willform a structure in which each branch of the polymer is itself abranched polymer. Either initiator cb*, corresponding to the sticky endand first complementary region of E1, or d*e*, corresponding the stickyend and first complementary region of Q1, will activate the chainreaction.

While non-linear amplification systems provide enhanced sensitivity overa linear system, they may also have an increased chance for spuriousinitiation of HCR and a resultant increase in false-positive signals.Several methods may be used to decrease the possibility for initiationof the system in the absence of the initiator. In systems utilizinghairpin monomers, these may include helix clamping, helix elongation andloop entropy ratchets.

The quadratic and exponential growth HCR schemes illustrated in FIGS. 2and 3 include long single-stranded regions (b* and e* respectively).These long regions could potentially function as weak initiators.Several methods are available to reduce spurious monomer polymerizationin the absence of initiator for both higher order growth schemes andlinear growth schemes. These include helix clamping, helix lengtheningand loop entropy ratchets. In helix clamping, the single strandedregions in one or more of the monomers are truncated at each end so thatthe helixes that they could potentially invade in other monomers areeffectively clamped at the ends by bases that are not present in thesingle stranded (b* and e*) regions. Experiments have shown that thiscan eliminate any spurious initiation. The amount of truncation that iseffective to decrease or eliminate spurious initiation can be determinedby routine experimentation. For example, control experiments can beperformed using fluorescent gel electrophoresis time courses to monitorstrand exchange between single stranded DNA and duplex DNA (e.g., strandb* invading duplex bb*) for different clamp lengths. Using spectrallydistinct dyes for the initially single stranded DNA and for the two DNAspecies in the duplex allows independent monitoring of all species asstrand exchange proceeds. These controls can provide a systematic basisfor section of clamp dimensions.

The length of the helices in the linear HCR scheme illustrated in FIG. 1contributes directly to the height of the kinetic barrier that preventsspurious polymerization between the two hairpin species. Interactionsbetween H1 and H2 are sterically impeded by the loop size. However, thelong helices (bb*) in each hairpin provide a more fundamental kineticbarrier; the length of the helices has a direct effect on the height ofthe kinetic barrier that impedes spurious HCR. An increase in the lengthof the helices will increase the initial kinetic barrier in theuninitiated system. Thus, in some embodiments utilizing hairpinmonomers, for example if spurious initiation is observed, the length ofthe duplex region can be increased to reduce the background noise. Thehelix length necessary to reduce polymerization in the absence ofinitiator to an acceptable level can be readily determined by routineexperimentation. In some embodiments helix lengthening is combined withhelix clamping.

In still other embodiments utilizing hairpin monomers, loop entropyratchets are used to reduce HCR in the absence of initiator. Aninitiator opens an HCR hairpin via a three-way branch migration. Thisreaction is reversible because the displaced strand is tethered in theproximity of the new helix. However, by increasing the length of thesingle-stranded loop, the entropy penalty associated with closing theloop increases. As a result, a longer loop will bias the reaction toproceed forward rather than returning to the uninitiated state. However,larger loops are more susceptible to strand invasion. To counter thiseffect and allow the use of larger loops, mismatches can be introducedbetween the loop sequences and the complementary regions of the othermonomers. Again, the loop length and amount of mismatch that producesthe desired reduction in non-specific HCR can be determined by theskilled artisan through routine experimentation.

Initiator

The initiator is preferably a nucleic acid molecule. The initiatorcomprises an initiator region that is complementary to a portion of anHCR monomer, preferably a portion of the monomer that is available forhybridization with the initiator while the monomer is in its kineticallystable state. The initiator also preferably comprises a sequence that iscomplementary to a portion of the monomer adjacent to the sticky endsuch that hybridization of the initiator to the sticky end causes aconformational change in the monomer and begins the HCR chain reaction.For example, the initiator may comprise a region that is complementaryto the first complementary region of the HCR monomer, as describedabove.

In the preferred embodiments, the sequence of the initiator iscomplementary the sticky end (initiator complementary region) and firstcomplementary region of a first monomer. As described above, in someembodiments this will also influence the sequence of the secondcomplementary region and the loop of the second monomer species.

In some embodiments the initiator is a nucleic acid that is to bedetected in a sample or a portion of a nucleic acid that is to bedetected. In this case, the sequence of the target nucleic acid is takeninto consideration in designing the HCR monomers. For example, theinitiator complement region, preferably a sticky end, of one monomer isdesigned to be complementary to a portion of the target nucleic acidsequence. Similarly, a region adjacent to the sticky end of the samemonomer can be designed to be complementary to a second region of thetarget sequence as well. Because the second monomer will hybridize tothe first monomer, the sequence of the second monomer will also reflectat least a portion of the sequence of the target nucleic acid.

In other embodiments, the initiator comprises at least a portion of anucleic acid that is part of a “initiation trigger” such that theinitiator is made available when a predetermined physical event occurs.In the preferred embodiments that predetermined event is the presence ofan analyte of interest. However, in other embodiments the predeterminedevent may be any physical process that exposes the initiator. Forexample, and without limitation, the initiator may be exposed as aresult of a change in temperature, pH, the magnetic field, orconductivity. In each of these embodiments the initiator is preferablyassociated with a molecule that is responsive to the physical process.Thus, the initiator and the associated molecule together form theinitiation trigger. For example, the initiator may be associated with amolecule that undergoes a conformational change in response to thephysical process. The conformational change would expose the initiatorand thereby stimulate polymerization of the HCR monomers. In otherembodiments, however, the initiation trigger comprises a single nucleicacid. The initiator region of the nucleic acid is made available inresponse to a physical change. For example, the conformation of theinitiation trigger may change in response to pH to expose the initiatorregion.

The structure of the trigger is preferably such that when the analyte ofinterest is not present (or the other physical event has not occurred),the initiator is not available to hybridize with the sticky end of amonomer. Analyte frees the initiator such that it can interact with ametastable monomer, triggering the HCR polymerization reactionsdescribed above. In some embodiments analyte causes a conformationalchange in the trigger that allows the initiator to interact with themonomer.

The initiator may be part of a trigger comprising a nucleic acid that islinked to or associated with a recognition molecule, such as an aptamer,that is capable of interacting with an analyte of interest. The triggeris designed such that when the analyte of interest interacts with therecognition molecule, the initiator is able to stimulate HCR.Preferably, the recognition molecule is one that is capable of bindingthe analyte of interest.

Recognition molecules include, without limitation, polypeptides, such asantibodies and antibody fragments, nucleic acids, such as aptamers, andsmall molecules. The use of an initiator bound to an aptamer isdescribed in more detail below.

In some particular embodiments, amplification of diverse recognitionevents is achieved by coupling HCR to nucleic acid aptamer triggers. Anaptamer is identified that is able to specifically bind an analyte ofinterest. The analyte is not limited to a nucleic acid but may be, forexample, a polypeptide or small molecule. The aptamer is linked to anucleic acid comprising an initiator region in such a way that theinitiator is unavailable to stimulate HCR in the absence of analytebinding to the aptamer.

Preferably, conformational changes in the aptamer secondary structureexpose the initiator segment. In one embodiment, such an aptamer triggeris a hairpin nucleic acid that comprises an initiator segment that iscomplementary to the initiator complement region or sticky end of an HCRmonomer. The aptamer trigger also comprises a complementary region thatis complementary to a region of the HCR monomer adjacent to the stickyend, a loop region and an aptamer sequence. The hairpin aptamer triggermay also comprise a region that enhances the stability of the hairpin inthe absence of aptamer binding to the analyte, such as a nucleic acidregion in one arm of the hairpin that is complementary to a region ofthe other arm.

FIG. 5A depicts a scheme for HCR amplification of ATP binding using anaptamer construct that exposes an initiator strand upon ATP binding. Thesticky end can act as a trigger for the HCR mechanism of FIG. 1 byopening hairpin H2. The region x is introduced to help stabilize thetrigger in the absence of analyte. The region b* includes both thehairpin loop and the portion of the stem complementary to x. Thistrigger mechanism is based on conformational changes in the aptamersecondary structure (Yingfu Li (2003) Journal of the American ChemicalSociety 125:4771-4778) that make the initiator strand available tostimulate HCR. FIG. 5B illustrates successful detection of ATP, as wellas specificity in differentiating ATP from GTP, as discussed in moredetail in the Examples below.

Detecting HCR

The products of HCR are readily detectable by methods known to one ofskill in the art for the detection of nucleic acids, including, forexample, agarose gel electrophoresis, polyacrylamide gelelectrophoresis, capillary electrophoresis, and gel-filled capillaryelectrophoresis. As the polymers comprise nucleic acids, they can bevisualized by standard techniques, such as staining with ethidiumbromide. Other methods also may be suitable including light scatteringspectroscopy, such as dynamic light scattering (DLS), viscositymeasurement, colorimetric systems and fluroscence spectropscopy.

In some embodiments HCR is monitored by fluorescence resonance energytransfer (FRET). Certain monomers are labeled with fluorescent dyes sothat conformational changes resulting from HCR can be monitored bydetecting changes in fluorescence. In one embodiment, one of a pair ofhairpin molecules is labeled with a fluorophore at the junction of theregion complementary to the initiator strand and the duplex region andlabeled at the opposing side of the duplex region with a quenchermolecule. Upon polymerization, the fluorophore and quencher areseparated spatially in the aggregated nucleic acid structure, providingrelief of the fluorescence quenching. In this case, the presence of asingle initiator is amplified by the chain of fluorescent events causedby HCR.

Because the size of the HCR products is inversely related to the amountof the target analyte in a sample, HCR can be used to determine analyteconcentration. The average molecular weight of the HCR products isobtained by standard measurements. Is some embodiments the averagemolecular weight of HCR products obtained from one sample is compared tothe average molecular weight of HCR products from one or more othersamples with an unknown analyte concentration. In this way, the relativeconcentration of analyte in each sample can be determined.

In other embodiments, the concentration of analyte is determined bycomparing the average molecular weight from a sample with unknownconcentration to the average molecular weight of HCR products from HCRreactions in one or more control samples with a known concentration ofthe analyte. In this way the concentration of analyte in the unknownsamples can be determined to be the same as one of the control samples,greater than one of the control samples, less than one of the controlsamples, or in the rance of concentration between two control samples.Thus, the number of control reactions can be adjusted based on theparticular circumstances to provide more or less sensitive determinationof analyte concentration. For example, if a relatively exact analyteconcentration is necessary, the number of control samples can beincreased. On the other hand, if only a broad idea of analyteconcentration is necessary, fewer control samples can be used.

Applications

HCR may be used to produce and amplifying a signal indicating thepresence of a target molecule. The initial signal can be the presence ofa target molecule comprising an initiator nucleic acid region. In otherembodiments the initial signal can be generated by any event thatexposes an initiator nucleic acid strand (for example, an aptamerbinding to its target ligand so as to expose an initiator strand, asdiscussed above). In both cases, hybridization of the initiator strandto the initiator complementary region on an HCR monomer triggerspolymerization of the HCR monomers. For some amplification applicationsin which detection is the primary objective, HCR may serve as anattractive alternative to PCR (polymerase chain reaction).

HCR can be used to detect a physical change in a sample. For example, asdiscussed above an initiator trigger may be used to triggerpolymerization of two or more HCR monomers in response to a physicalchange in the sample. This may be, for example, and without limitation,a change in pH, temperature, magnetic field or conductivity in thesample.

In other embodiments HCR is used to identify a physical characteristicof a sample. An initiator trigger may be used that is activated onlywhen the sample has a particular characteristic, such as a particularpH, temperature or conductivity. In one embodiment an initiator triggeris utilized that exposes the initiator and triggers HCR only when the pHis above a predetermined level. A sample can then be contacted with HCRmonomers and the initiator trigger. After incubation, the sample isanalyzed for the presence of HCR reaction products. The presence of suchproducts indicates a pH above the predetermined level.

HCR may be used to construct a biosensor. In one embodiment the sensorwould comprise two or more HCR monomers and would detect the presence ofinitiator in a sample. Whenever initiator is present, HCR will occur,resulting in the creation of a polymer from the individual monomerspecies.

HCR can be used to identify the presence of nucleic acids of interest ina sample. For example, nucleic acids associated with a pathogen can beidentified in a biological sample obtained from a patient. In someembodiments, a sample is obtained from a patient and tested for thepresence of nucleic acids associated with a virus, such as HIV. One ormore pairs of HCR monomers are contacted with the sample, where at leastone of the monomers comprises an initiator complementary region that iscomplementary to a portion of the nucleic acid to be detected. Afterincubation, the sample is tested for the presence of polymerized HCRmonomers, such as by gel electrophoresis.

Furthermore, the average molecular weight of the resulting polymers willbe inversely related to the initiator concentration. As a result, HCRcan be used for quantitative sensing. In one embodiment theconcentration of analyte in a sample is determined by comparing HCRproducts in the sample with HCR products obtained from control reactionsusing known concentrations of analyte.

For some applications, it may be useful to employ HCR for bothamplification and capture of a target. Thus, the HCR mechanism can bedesigned to capture a target molecule, leaving the target tethered tothe resulting polymer. The separation step is facilitated by theincreased size of the HCR product with respect to the analyte ofinterest. Thus, even small, rare nucleic acid targets can be readilyisolated from a sample. In one embodiment the HCR reactions are run on agel under conditions that do not disrupt binding between the target andthe HCR monomer. The HCR products are then isolated from the gel and thetarget is purified.

More sophisticated biosensors can be created by the incorporation of DNAor RNA aptamers or other mechanisms that expose HCR initiator strandsonly in the presence of a particular analyte of interest or a particularphysical change in the system. FIG. 5A shows how ATP can be used totrigger HCR. FIG. 5B shows illustrative results demonstratingsensitivity to initiator concentration and specificity in detecting ATPrelative to GTP.

Thus in some embodiments an initiator region is linked to a recognitionmolecule that is specific for an analyte to be detected in a sample toform an initiator trigger. The initiator is only made available tostimulate HCR upon analyte binding to the recognition molecule.Preferably, the recognition molecule is an aptamer that is specific foran analyte of interest, such as a polypeptide associated with a diseaseor disorder. The polypeptide may be, for example, a cancer antigen or apolypeptide associated with a pathogen or infectious agent.

In one embodiment a biological sample, such as blood, urine,cerebrospinal fluid, or tissue, is obtained from a patient suspected ofsuffering from a disease or disorder. The sample is incubated with oneor more pairs of HCR monomers and an initiator trigger moleculecomprising an initiator and an aptamer specific for a polypeptideassociated with the disease or disorder. Following incubation the sampleis analyzed for the presence of polymers comprising the HCR monomers.The presence of the polymers indicates that the analyte associated withthe disease or disorder is present in the sample.

EXAMPLES

HCR monomers comprising DNA sequences were designed using a combinationof criteria (Dirks et al. Nucleic Acids Research 32:1392-1403 (2004)).These included sequence symmetry minimization (Seeman N.C. J. Theor.Biol. 99:237-247 (1982)), the probability of adopting the targetsecondary structure at equilibrium (Hofacker et al. Monatsh. Chem.125:167-188 (1994)), the average number of incorrect nucleotides atequilibrium relative to the target structure (Dirks et al. Nucleic AcidsResearch 32:1392-1403 (2004)) and hybridization kinetics (Flamm et al.RNA 6:325-338 (2000)). The sequences of the monomers and initiator forthe basic HCR system illustrated in FIG. 1 and the aptamer trigger HCRsystem illustrated in FIG. 5 are shown in Table 1. The aptamer systemincluded new sequences to ensure compatibility with the fixed sequenceof the aptamer. DNA was synthesized and purified by Integrated DNATechnologies (Coralville, Iowa). TABLE 1 System Strand Sequence* BasicH1 5′- TTA ACC CAC GCC GAA TCC TAG ACT CAA AGT AGT CTA (SEQ ID NO: 1)GGA TTC GGC GTG - 3′ H2 5′- AGT CTA GGA TTC GGC GTG GGT TAA CAC GCC GAATCC (SEQ ID NO:2) TAG ACT ACT TTG - 3′ I 5′- AGT CTA GGA TTC GGC GTG GGTTAA - 3′ (SEQ ID NO: 3) Aptamer† H1 5′- CAT CTC GGT TTG GCT TTC TTG TTACCC AGG TAA CAA (SEQ ID NO: 4) GAA AGC CAA ACC - 3′ H2 5′- TAA CAA GAAAGC CAA ACC GAG ATG GGT TTG GCT TTC (SEQ ID NO: 5) TTG TTA CCT GGG - 3′I^(ATP) 5′- CCC AGG TAA CAA GAA AGC CAA ACC TCT TGT TAG CTG (SEQ ID NO:6) GGG GAG TAT TGC GGA GGA AGG T - 3′ I 5′- CCC AGG TAA CAA GAA AGC CAAACC - 3′ (SEQ ID NO: 7)*In the hairpin sequences, loops are in bold and sticky ends areitalicized.†Aptamer nucleotides are italicized and bolded.

For the basic HCR system illustrated in FIG. 1, concentrated DNA stocksolutions were prepared in buffer that was later diluted to reactionconditions. The buffer comprised 50 mM Na2HPO4/0.5M NaCl (pH 6.8).

Monomers H1 and H2 (FIG. 1B) were mixed at various concentrations in thepresence and absence of initiator. Stock solutions of I, H1 and H2 werediluted in reaction buffer to three times their final concentration and9 μl of each species was combined, in that order to give a 27 μlreaction volume. Six different concentrations of initiator were used(0.00, 10.00, 3.20, 1.00, 0.32 and 0.10 μM) in a 1 μM mixture of H1 andH2. Reactions were incubated at room temperature for 24 hours beforerunning 24 μl of each product on a gel. Samples were loaded on 1%agarose gels containing 0.5 μg EtBr per ml of gel volume. The gels wereprepared using 1×SB buffer (10 mM NaOH, pH adjusted to 8.5 with boricacid). The agarose gels were run at 150 V for 60 minutes and visualizedunder UV light.

FIG. 1D illustrates the results of the HCR reactions and the effect ofinitiator concentration on amplification. Lanes 2-7 of the gel shown inFIG. 1D are the results of the HCR reactions at the various initiatorconcentrations, respectively. Lanes 1 and 8 are DNA markers with 100-bpand 500-bp increments respectively. As illustrated by FIG. 1D,introduction of an initiator strand triggers a chain reaction ofhybridization that results in the production of polymers of varioussizes. The average molecular weight of the polymers is inversely relatedto the initiator concentration (FIG. 1(d)). The inverse relationshipfollows from the fixed supply of monomer hairpins, but the phenomenonwas observed after 10 minutes, when the supply of monomers had not beenexhausted.

These results confirmed an earlier experiment in which 1 μM of H1 and H2were reacted overnight in 0.5M NaCl, 50 mM Na₂HPO₄ at pH 6.5 withinitiator at concentrations of 0, 1, 0.1, 0.01, 0.001 and 0.0001 μM.With no initiator HCR reactions were not observed. In addition, novisible polymer growth was observed at initiator concentrations of 0.001and 0.0001 μM. At the other initiator concentrations an inverserelationship was observed between the initiator concentration and theaverage molecular weight of the resulting polymers.

In another set of experiments, the aptamer trigger illustrated in FIG.5A was utilized. The aptamer trigger (I^(ATP)) comprised an initiatorregion corresponding to the initiator used in the experiments describedabove, linked to an aptamer that is able to specifically interact withATP (Huizenga et al. Biochemistry 34:656-665 (1995)). In addition, theaptamer trigger comprises a stabilizing region designed to stabilize thetrigger in the absence of ATP. The aptamer trigger is designed such thatin the presence of ATP the hairpin is opened and the initiator regionexposed, thereby triggering polymerization of the H1 and H2 monomers.The sequence of I^(ATP) is provided in Table 1, above.

Reactions were carried out with various combinations of H1, H2, I,I^(ATP), ATP and GTP. Concentrated stock solutions of the constituentswere diluted to reaction conditions: 5 mM MgCl2/0.3 M NaCl/20 mM Tris(pH 7.6). Reactions were performed with 1.4 mM ATP and/or GTP. DNAspecies were combined to yield 1 μM concentrations in 27 μl of reactionbuffer, with additions made in the following order: buffer and/orinitiator I or aptamer trigger I^(ATP), H1, and then H2 (I and I^(ATP)interact with H2 rather than H1). In this case, 1 μl of 40 mM ATP, 40 mMGTP or water was added to each reaction, as appropriate, for a totalreaction volume of 28 μl.

Reactions were incubated at room temperature for one hour and run onagarose gels (as described above) or native polyacrylamide gels. Nativepolyacrylamide gels were 10% precast gels made with 1×TBE buffer (90 mMTris, 89 mM boric acid, 2.0 mM EDTA, pH 8.0). The polyacrylamide gelswere run at 150V for 40 minutes in 1×TBE and stained for 30 minutes in asolution containing 5 μg EtBr per ml.

FIG. 5B shows a representative agarose gel illustrating that the aptamertrigger I^(ATP) can initiate polymerization of H1 and H2 in the presenceof ATP and that ATP can be distinguished from GTP. Hairpins H1 and H2did not polymerize when mixed in the absence of initiator (H1+H2; Lane1), but did polymerize when the initiator I was added (H1+H2+I; Lane 2).ATP alone was unable to trigger the polymerization of the hairpinmonomers (H1+H2+ATP; Lane 3) and no polymers were observed from thecombination of aptamer trigger with ATP in the absence of hairpinmonomers (IATP+ATP; Lane 4). Some weak spurious HCR was observed in theabsence of ATP from the combination of monomers and aptamer trigger(H1+H2+I^(ATP); Lanes 5) or in the presence of GTP (H1+H2+I^(ATP)+GTP;Lane 7), respectively. Strong HCR amplification of ATP recognition wasseen when the monomers were combined with the aptamer trigger in thepresence of ATP (H1+H2+I^(ATP)+ATP; Lane 6). A DNA ladder is shown inLane 8 (100-1000 bp in 100 bp increments).

The kinetics of HCR reactions were explored using fluorescencequenching. The adenine analog 2-aminopurine (2AP) fluoresces when singlestranded but is significantly quenched when in a stacked double-helicalconformation (Rachofsky et al. Biochemistry 40: 996-956 (2001)). Monomerusage was monitored as polymerization occurred by replacing H1 with thelabeled hairpin H1^(2AP). H1^(2AP) was prepared by substituting 2AP forthe third base (A) in the sticky end of H1 (see Table 1). Monitoring 2APfluorescence was used rather than standard end-labeled strands becausethe local environment of quenched 2AP was the same regardless of whetherinitiator (I) or monomer (H2) performs the quenching.

Fluorescence data were obtained using a fluorometer from PhotonTechnology International (Lawrenceville, N.J.), with the temperaturecontroller set to 22° C. Excitation and emission wavelengths were 303and 365 nm, respectively, with 4 nm bandwidths. Stock solutions of 0.40μM H12AP and 0.48 μM H2 were prepared in reaction buffer as describedabove, heated to 90° C. for 90 seconds and allowed to cool to roomtemperature for 1 hour before use. For each experiment, 250 μl of H12APwas added to either 250 μl of H2 or 250 μl of reaction buffer. These0.20 μM H12AP solutions were allowed to sit at room temperature for atleast 24 hours before fluorescence measurements were taken. The initialsignal was obtained after rapidly pipetting the sample in the cuvette toobtain a stable fluorescence baseline. After acquiring at least 2,000seconds of the baseline, runs were paused for about 1 minute to add 20μl of initiator (either 20 μM or 2.5 μM) and allow mixing by rapidpipetting. The final reaction volume was 520 μl for all experiments. Thevariation in initial fluorescence intensities was about 10% across threeexperiments.

As evidenced by the results presented in FIG. 1E the hairpin monomers H1and H2 do not hybridize in the absence of initiator. Addition ofinitiator (I) to the hairpin mixture led to fluorescence quenching viaHCR (bottom band from 0 to 2000 seconds, then dropping to middle bandfrom 2000 seconds on)

The same quenched baseline was achieved without HCR by combiningH1^(2AP) with excess initiator in the absence of H2 (FIG. 1(E), middleband from 0 to 2000 seconds, then dropping to bottom band from 2000seconds on). In this case, each initiator 2AP molecule caused onefluorescent signaling event by binding to H1^(2AP). With H2 present, HCRperformed fluorescent amplification, allowing each initiator molecule toalter the fluorescence of multiple hairpins.

Addition of insufficient initiator to H1^(2AP) provided only partialquenching (FIG. 1(E), top band), demonstrating that HCR, and notinitiator alone, was responsible for exhausting the supply of H1^(2AP)monomer.

Although the foregoing invention has been described in terms of certainpreferred embodiments, other embodiments will be apparent to those ofordinary skill in the art. Additionally, other combinations, omissions,substitutions and modification will be apparent to the skilled artisan,in view of the disclosure herein. Accordingly, the present invention isnot intended to be limited by the recitation of the preferredembodiments, but is instead to be defined by reference to the appendedclaims. All references cited herein are incorporated by reference intheir entirety.

1. A method for detecting an analyte in a sample by hybridization chainreaction, the method comprising: contacting the sample with a firstmetastable monomer comprising an initiator complement region; contactingthe sample with a second metastable monomer comprising a regioncomplementary to a portion of the first monomer; and identifyingpolymers comprising the first and second monomers, wherein the first andsecond monomers polymerize in the presence of an initiator.
 2. Themethod of claim 1, wherein the initiator is a nucleic acid.
 3. Themethod of claim 1, wherein the analyte comprises the initiator.
 4. Themethod of claim 1, wherein polymerization of the first and secondmonomers is initiated by hybridization of the initiator to the initiatorcomplement region of the first monomer.
 5. The method of claim 1,wherein the initiator is able to hybridize with the initiator complementregion when analyte is present in the sample.
 6. The method of claim 1,wherein the sample is additionally contacted with an initiation trigger.7. The method of claim 6, wherein the initiation trigger comprises anaptamer.
 8. The method of claim 7, wherein the aptamer is able tospecifically bind the analyte.
 9. The method of claim 7, wherein theinitiation trigger comprises the initiator.
 10. The method of claim 9,wherein the initiator is able to hybridize to the first monomer when theaptamer is bound by the analyte.
 11. The method of claim 1, whereinidentifying polymers comprises gel electrophoresis.
 12. The method ofclaim 1, wherein at least one of the first and second monomers isfluorescently labeled.
 13. The method of claim 1, wherein the analyte isa nucleic acid associated with a pathogen.
 14. The method of claim 1,wherein the sample is obtained from a patient.
 15. The method of claim1, additionally comprising contacting the sample with a third and fourthmetastable monomers.
 16. The method of claim 1, wherein the first andsecond metastable monomers are hairpin monomers.
 17. A method of forminga structure comprising hybridized nucleic acid monomers, the methodcomprising: a) providing an initiator comprising a nucleic acidinitiation region; b) providing a first nucleic acid hairpin monomercomprising a duplex region, a first loop region, and an initiatorcomplement region that is substantially complementary to the initiationregion; (c) providing a second nucleic acid hairpin monomer comprising asecond duplex region, and a propagation region that is substantiallycomplementary to the initiator complement region of the first monomer;wherein the first and second monomers hybridize in a chain reaction uponbinding of the initiator to the first monomer.
 18. The method of claim17, wherein the first loop region comprises from about 3 to about 30nucleotides.
 19. The method of claim 17, wherein the propagation regionof the second monomer is a second loop region.
 20. The method of claim17, wherein the initiator is an analyte to be detected in a sample. 21.The method of claim 17, wherein the initiator is a single strandednucleic acid.
 22. The method of claim 17, wherein the initiatoradditionally comprises a recognition molecule.
 23. The method of claim22, wherein the recognition molecule is an aptamer.
 24. The method ofclaim 17, wherein the initiator undergoes a conformational change in thepresence of an analyte.
 25. The method of claim 25, wherein the analytecomprises a polypeptide
 26. The method of claim 17, wherein thestructure comprising hybridized monomers is detected by a methodselected from the group consisting of gel electrophoresis, capillaryelectrophoresis, mass spectrometry, light scattering spectroscopy,colorimetry and fluorescent spectroscopy.
 27. The method of claim 17,further comprising: providing a third nucleic acid monomer comprising afirst complementary region that is complementary to a portion of thefirst and/or second monomer; and providing a fourth nucleic acid monomercomprising a second complementary region that is complementary to aportion of the third monomer.
 28. The method of claim 27, wherein thefirst monomer comprises a bulge region that is complementary to aportion of the third monomer.
 29. An kit for the detection of an analytein a sample, the kit comprising: a) a first metastable nucleic acidmonomer comprising an initiator complement region; and b) a secondmetastable nucleic acid monomer comprising a propagation region that issubstantially complementary to a portion of the first nucleic acid. 30.The kit of claim 29, wherein the first and second monomers are hairpinmonomers.
 31. The kit of claim 29, additionally comprising a third andfourth metastable nucleic acid monomer.
 32. The kit of claim 29, whereinthe initiation complement region is substantially complementary to aportion of the analyte to be detected.
 33. The kit of claim 29,additionally comprising an initiation trigger.
 34. The kit of claim 33,wherein a portion of the initiation trigger is substantiallycomplementary to the initiator complement region of the first monomer.35. The kit of claim 31, wherein the initiation trigger comprises anaptamer.
 36. The kit of claim 35, wherein the aptamer is specific forthe analyte to be identified.